It has been widely appreciated that biological systems resist environmental, stochastic, and genetic perturbations. This phenomenon has been termed homeostasis, buffering, robustness and canalization. It is becoming possible to investigate these concepts at a molecular level. Recent results indicate that molecular circuits are designed to minimize the phenotypic consequences of perturbation, a process we will term "buffering". We propose to develop paradigms and methods for understanding how genetic variation is buffered using the DNA replication and dNTP synthesis circuits of the yeast, Saccharomyces cerevisiae as test cases. Understanding such buffering mechanisms is important to three broad areas of biology: (1) how genetic variation, the material of evolution, is accumulated in a species; (2) which genes influence the phenotypic expression of genetic variability, for example in the highly variable presentation of human disease in different individuals; and (3) the design principles of biological systems. Our specific aims are the following: 1. Design and employ methods to control the quantity of specific essential proteins in the DNA replication machinery and dNTP biosynthesis pathway. 2. Comprehensively identify the nonessential proteins that influence the capacity of the cell to withstand reduced expression of key essential proteins. 3. Compare the buffering capacity contributed by specific proteins quantitatively and assign them to different epistasis groups. 4. Explore differences among natural isolates of S. cerevisiae in their buffering mechanisms.